This application claims the benefit of foreign priority to European Application No. 99103869.6, filed on Mar. 1, 1999. This European priority document is incorporated by reference herein.
Objects of the present invention are low molecular weight peptide derivatives which are able to act as inhibitors of the interaction between laminin and nidogen (laminin/nidogen interaction), a process for their preparation, pharmaceutical compositions prepared therefrom and their use for preparing pharmaceuticals and for identifying inhibitors of the laminin/nidogen interaction.
The association of laminin (an 800 kDa glycoprotein) and nidogen (a 160 kDa glycoprotein) is regarded as a crucial biomolecular mechanism in the synthesis and stabilization of basement membranes (Mayer, U. and Timpl, R. (1994) in: Extracellular Matrix Assembly and Structure (P. D. Yurchenco, D. Birk and R. P. Mecham, Ed.) S. 389-416, Academic Press, Orlando, Fla.). The ability of nidogen to form ternary complexes with all main constituents of the basement membrane such as, for example, xcex31-containing laminin isoforms (for nomenclature see: Burgeson, R. E.; Chiquet, M.; Deutzmann, R.; Ekblom, P.; Engel, J.; Kleinmann, H.; Martin, G. R.; Meneguzzi, G.; Paulsson M.; Sanes, J.; Timpl, R.; Tryggvasson, K.; Yamada, Y.; Yurchenco, P. D. (1994) Matrix Biology 14; 209-211), collagen IV, perlecan and fibulin, and the association structures of each of them, means that it assumes the function of a linker which connects together, spatially organizes and stabilizes the independent macrostructures (Fox, J. W.; Mayer, U.; Nischt, R.; Aumailley, M.; Reinhardt, D.; Wiedemann, H.; Mann, K.; Timpl, R.; Krieg, T., Engel, J.; and Chu, M.-L. (1991) EMBO J. 10, 3137-3146).
Basement membranes are highly specialized extracellular structures which are attributed with important functions in the control of cell and tissue functions, tissue architecture, tissue interactions, cell growth, cell transformation, cell migration and in tissue-specific gene expression (Adams, J. C. and Waft, F. M. (1993) Development 117, 1183-1198).
Experiments with polyclonal antilaminin antibodies have provided clear evidence of the central function of the laminin/nidogen interaction in the synthesis of a functional basement membrane. The described antibodies were obtained by immunizing rabbits with laminin P1 or with the recombinantly produced nidogen-binding domain of laminin (xcex31 III 3-5). The antibodies concentrated by affinity chromatography on laminin P1 or laminin xcex31 III 3-5 matrices showed complete inhibition of the laminin/nidogen association in inhibition assays. However, this is based on steric blockade of the access of nidogen to laminin by the antibodies, whose binding regions are located in the vicinity of the nidogen-binding sequences of laminin (Mayer, U.; Nischt, R.; Pxc3x6schl, E.; Mann, K.; Fukuda, K.; Gerl, M.; Yamada, Y.; Timpl, R. (1993) EMBO J. 12; 1879-1885).
In embryonic organ cultures, the described antibodies inhibited both the development of renal tubules, the formation of pulmonary alveoli and the morphogenesis of the embryonic salivary gland. These three models are representative of ontogenesis programs which depend on unimpeded synthesis of new basement membrane (Ekblom, P.; Ekblom, M.; Fecker, L.; Klein, G.; Zhang, H.-Y.; Kadoya, Y.; Chu, M.-L.; Mayer, U.; Timpl, R. (1994) Development 120; 2003-2014).
Antibodies directed against the laminin xcex31 chain sequence region which is essential for nidogen binding are likewise able to inhibit the laminin/nidogen association. The inhibition is, however, competitive, in contrast to the antilaminin antibodies described above, because they compete directly with the nidogen for the binding site on laminin (WO 98/31709).
A monoclonal antibody of the IgM subclass (antilaminin P1 A6/2/4-DSM ACC2327; see WO 98/31709) inhibits the laminin/nidogen interaction in vitro with an IC50 of 30 nM. Like the polyclonal antilaminin antibody preparation described above, it prevents the morphogenesis of the embryonic salivary gland in organ culture. This underlines the specificity of the laminin/nidogen interaction, and the importance of the LE-4 module and of the identified sequence region in the laminin xcex31 III 4 domain in this interaction.
The nidogen binding domain of laminin has been unambiguously identified and characterized in terms of its location, sequence and its spatial structure (X-ray crystal structure and NMR structure) (Gerl, M.; Mann, K.; Aumailley, M.; Timpl, R. (1991) Eur. J. Biochem. 202; 167-174. Mayer, U.; Nischt, R.; Pxc3x6schl, E.; Mann, K.; Fukuda, K.; Gerl, M.; Yamada, Y.; Timpl, R. (1993) EMBO J. 12; 1879-1885. Baumgartner, R.; Czisch, M.; Mayer, U.; Pxc3x6schl, E.; Huber, R.; Timpl, R.; Holak, T. A. (1996) J. Mol. Biol. 257; 658-668. Stetefeld, J.; Mayer, U.; Timpl, R.; Huber, R. (1996) J. Mol. Biol. 257; 644-657). It is located in an xe2x80x9cLE modulexe2x80x9d (laminin type epidermal growth factor-like) of the short arm of the xcex31 chain of laminin, in the domain xcex31 III 4. xe2x80x9cLE modulesxe2x80x9d are structural motifs of 50-60 amino acids which have a complex folding pattern, analogous to EGF, with 4 disulfide bridges (Bairoch, A.; (1995) Nomenclature of extracellular domains. The SWISS-PROT Protein sequence data bank. release 310. Engel, J. (1989) FEBS Letters 251; 1-7).
High-affinity binding of nidogen to the complementary laminin domain has been detected for laminin P1 from the EHS tumor of mice, laminin 2 and laminin 4 from human placenta and laminin from drosophila. The cause of this species-overlapping binding specificity is the extremely large identity of sequences present in the xcex31 III 4 domain for the species investigated. It is 97% between human and mouse, 61% between mouse and drosophila and, astonishingly, 51% between mouse and Caenorhabditis elegans when the whole domain is taken into account (Pikkarinen, T.; Kallunki, T.; Tryggvasson, K. (1987) J. Biol. Chem. 263; 6751-6758. Chi, H.-C.; Hui, C.-F. (1989) J. Biol. Chem. 264; 1543-1550. Wilson, R. et al.(1994) Nature 368: 32-38. Pxc3x6schl, E.; Mayer, U.; Stetefeld, J.; Baumgartner, R.; Holak, T. A.; Huber, R.; Timpl, R. (1996) EMBO J. 15: 5154-5159).
Besides the dependency of nidogen binding on an intact three-dimensional structure, unambiguous sequence regions located in the Sxe2x80x94S stabilized loops a and c of the domain xcex31 III 4 have been identified. Five essential amino acids have been identified, four located inside a section of 7 amino acids in loop a, and a tyrosine side-chain in loop c (Mann, K.; Deutzmann, R.; Timpl, R. (1988) Eur. J. Biochem. 178; 71-80).
Synthetic peptides which can be derived from the appropriate regions of the xcex31 III 4 domain and are able to inhibit completely the laminin/nidogen binding in specific binding assays have been disclosed by J. W. Fox and R. Timpl (U.S. Pat. No. 5,493,008).
The high-affinity binding to the laminin binding site of nidogen is thought to require an interaction with a tyrosine or histidine from a loop (loop c) adjacent to the actual binding sequence. This aromatic interaction was postulated as a precondition for inhibition in the IC50 range  less than 500 nM on the basis of the 3D structure of the laminin xcex31 III 3-5 and as a result of the structure/function relations described in the U.S. Pat. No. 5,493,008. The question of whether loop c interacts directly with the nidogen, or whether it makes a contribution to stabilizing the suitable spatial structure of the NIDPNAV (SEQ ID NO:1) sequence region remained unclarified, however (Pxc3x6schl, E.; Fox, J. W.; Block, D.; Mayer, U.; Timpl, R, (1994) EMBO J. 13; 3741-3747. Baumgartner, R.; Czisch, M.; Mayer, U.; Pxc3x6schl, E.; Huber, R.; Timpl, R.; Holak, T. A. (1996) J. Mol. Biol. 257; 658-668. Stetefeld, J.; Mayer, U.; Timpl, R.; Huber, R. (1996) J. Mol. Biol. 257; 644-657).
The laminin/nidogen interaction is influenced by a strong conformational component (Mayer, U.; Nischt, R.; Pxc3x6schl, E.; Mann, K.; Fukuda, K.; Gerl, M.; Yamada, Y.; Timpl, R. (1993) EMBO J. 12; 1879-1885. Mann, K.; Deutzmann, R.; Timpl, R. (1988) Eur. J. Biochem. 178; 71-80). The synthetic peptides which can be derived from the nidogen binding site of laminin are not able to form a disulfide linkage pattern as is present in LE modules, but they show an activity in inhibition assays which is about 400-10,000-fold weaker than that of intact laminin P1 or laminin xcex31 III 3-5 (Pxc3x6schl, E.; Fox, J. W.; Block, D., Mayer, U.; Timpl, R, (1994) EMBO J. 13; 3741-3747. J. W. Fox and R. Timpl; U.S. Pat. No. 5,493,008). This decline in activity is not unusual, since it is known that peptides may assume a myriad of different conformations in aqueous solution and that only a certain percentage of peptides is to be found in the biologically active conformation. The most active peptide described to date (IC50 of 22 nM) has a molecular weight of about 2700 Da (≅about 50% of an LE module). It comprises an intact Sxe2x80x94S loop which presumably stabilizes the structure of the essential NIDPNAV (SEQ ID NO:1) sequence region (Pxc3x6schl, E.; Fox, J. W.; Block, D.; Mayer, U.; Timpl, R, (1994) EMBO J. 13; 3741-3747. J. W. Fox and R. Timpl; U.S. Pat. No. 5,493,008).
The chemical formula of the sequence NIDPNAV (Asn-lie-Asp-Pro-Asn-Ala-Val) (SEQ ID NO:1) is as follows: 
Inhibitors of the laminin/nidogen interaction should be suitable for preparing pharmaceuticals for diseases which are related to an increased or unwanted synthesis of basement membranes.
Such diseases are e.g. all types of late complications of diabetes which are accompanied by thickening of basement membranes (especially in the kidney, eye, vascular system), hepatic fibrosis, especially alcoholic hepatic fibrosis, characterized by synthesis of a continuous basement membrane in the sinusoids and a capillarization caused thereby, all fibroses (chronic or iatrogenic) in which an increased synthesis of basement membrane or components of the basement membrane can be observed (kidney, lung, skin), atherosclerosis characterized by a limitation of the regulation of lipid metabolism, which may be caused inter alia by impaired filtration of lipoproteins through the partly capillarized liver sinusoids (the pathological changes in the vascular system which can be observed with atherosclerosis may also in part be attributed to modifications of the composition and structure of the basement membranes in the vessels), diseases in which angiogenesis contributes to a deterioration in the clinical picture, for example cancers in which neovascularization is required for tumor growth, diabetic retinopathy, retrolental fibroplasia, disorders with a strong inflammatory component (for example rheumatoid arthritis, osteoarthritis, vasculitis), hemangiomas, psoriasis, and many others.
The use of peptides like those described in U.S. Pat. No. 5,493,008 as medicine is however limited to a considerable extent because of their conformational flexibility, their instability to proteases and their poor bioavailability and pharmacodynamics (Milner-White, E. J. (1989) Trends Pharmacol. Sci. 10; 70-74. Verber, D. F.; Freidinger, R. M.; (1985) Trends Neurosci. 8; 392-396. Hruby, V. J. (1994) in: Peptides, Proc. Thirteenth American Peptide Symposium; (Hodges, R. S.; Smith, J. A.; Ed.) S. 3-17; ESCOM: Leiden, Netherlands).
An object of this application was thus to find low molecular weight peptide derivatives which are able to interact specifically with the laminin binding site of nidogen and to inhibit competitively the association between laminin and nidogen at low concentration.
Therefore, an object of the present invention is a compound of the formula I 
wherein
R1 is a group of one of the following formulae 
xe2x80x83wherein
R4 means xe2x80x94A, xe2x80x94NH2, xe2x80x94NHR, xe2x80x94NR2, A2, xe2x80x94NHR1, 
and R5 means xe2x80x94(CH2)lCOOA, xe2x80x94(CH2)lCONH2, xe2x80x94(CH2)lNH2 or xe2x80x94(CH2)lxe2x80x94SO3H, 
and X is a group of one of the following formulae 
xe2x80x83wherein
Y means O, S, xe2x80x94N(A)xe2x80x94COxe2x80x94 or xe2x80x94(CH2)rxe2x80x94,
D means (CH2)r, O, S, NH, NR, (CH2)rxe2x80x94O, (CH2)rxe2x80x94S, (CH2)rxe2x80x94NH or (CH2)rNR and
R2 means xe2x80x94A, xe2x80x94Exe2x80x94OH, xe2x80x94Exe2x80x94COOH or xe2x80x94Exe2x80x94CONH2,
wherein E means a linear or branched C1-C10-alkyl chain, which is unsubstituted or substituted by xe2x80x94A, xe2x80x94(CH2)mxe2x80x94OH, xe2x80x94(CH2)mxe2x80x94COOH, xe2x80x94(CH2)mxe2x80x94C(O)NA2 or by a C5-C10-cycloalkyl group,
or E means C5-C10-cycloalkyl, which is unsubstituted or substituted by xe2x80x94A, xe2x80x94(CH2)mxe2x80x94OH, xe2x80x94(CH2)mxe2x80x94COOH, xe2x80x94(CH2)mxe2x80x94C(O)NA2 or by a C5-C10-cycloalkyl group,
and R3 is a group of one of the following formulae 
wherein R6 means xe2x80x94H, xe2x80x94COOH, xe2x80x94CONH2, xe2x80x94CONHR, xe2x80x94CONR2, xe2x80x94CH2OH or 
and wherein R7 means a linear or branched C1-C10-alkyl group, which is unsubstituted or substituted by xe2x80x94A, xe2x80x94(CH2)mxe2x80x94OH, xe2x80x94(CH2)mxe2x80x94COOH, xe2x80x94(CH2)mxe2x80x94C(O)NA2 or by a C5-C10-cycloalkyl group,
or R7 means a C5-C10-cycloalkyl group, which is unsubstituted or substituted by xe2x80x94A, xe2x80x94(CH2)mxe2x80x94OH, xe2x80x94(CH2)mxe2x80x94COOH, xe2x80x94(CH2)mxe2x80x94C(O)NA2 or by a C5-C10-cycloalkyl group,
and R means branched or unbranched C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkinyl, C5-C10-cycloalkyl, Het or Ar which are optionally substituted by one ore more halogen, C1-C6-alkyloxy, branched or unbranched C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkinyl or C5-C10-cycloalkyl groups or by xe2x80x94C1-C6-alkyl-Het, xe2x80x94C1-C6-alkyl-Ar, xe2x80x94Oxe2x80x94C1-C6-alkyl-Het, xe2x80x94Oxe2x80x94C1-C6-alkyl-Ar, Het or by Ar, wherein
Het means a monocyclic or bicyclic, 5- up to 10-membered aromatic or non-aromatic ring containing 1 or 2 equal or different hetero-atoms as members of said ring, selected from the group consisting of nitrogen, oxygen and sulfur,
xe2x80x83which is unsubstituted or substituted by one or more hydroxy or carboxy groups, and wherein
Ar means a monocyclic or bicyclic 5- up to 10-membered aromatic ring which is unsubstituted or substituted by one or more hydroxy or carboxy groups, and
Z means (CH2)m, O, S, NH, NR, Nxe2x80x94C(O)xe2x80x94R or NSO2R,
A means H or C1-C4-alkyl and
l, m and r are integers from 0 to 3,
n and k are integers from 1 to 2,
p is an integer from 0 to 1 and
q is an integer from 1 to 3,
in all its stereoisomeric forms and mixtures thereof in all ratios including all its physiologically tolerable salts.
Physiologically tolerable salts are for example salts of inorganic and organic acids, e.g. hydrochloric acid, sulfuric acid, acetic acid, citric acid or p-toluenesulfonic acid, or salts of inorganic and organic bases, such as NH4OH, NaOH, KOH, Ca(OH)2, Mg(OH)2, diethanolamine or ethylenediamine, or salts of amino acids, such as arginine, lysine, lysyl-lysine or glutamic acid.
One preferred embodiment of the present invention is a compound of formula I wherein n is 1.
A further preferred embodiment is a compound of formula I wherein R in group X means Het or Ar which are optionally substituted by xe2x80x94C1-C6-alkyl-Het, xe2x80x94C1-C6-alkyl-Ar, xe2x80x94Oxe2x80x94C1-C6-alkyl-Het, xe2x80x94Oxe2x80x94C1-C6-alkyl-Ar, Het or by Ar. More preferably, R in group X means Het. For example Het means 
A preferred embodiment of the present invention is also compound of formula I wherein R in group X means Ar which is optionally substituted by xe2x80x94C1-C6-alkyl-Ar, xe2x80x94Oxe2x80x94C1-C6-alkyl-Ar or by Ar. Preferably R in group X means Ar.
For example Ar means 
A preferred embodiment is also a compound of formula I wherein R in group X means 
In the compound of formula I X is preferably a group of the following formula: 
Preferably, Y means xe2x80x94(CH2)r, wherein r is preferably 0 or 1 and k is preferably 1 or 2.
A further preferred embodiment of the present invention is a compound of formula I wherein X is a group of the following formula 
wherein D preferably means xe2x80x94(CH2)rxe2x80x94, wherein r is 0 or 1.
An also preferred embodiment of the present invention compound of formula I wherein R1 is a group of the following formula 
wherein Z means preferably (CH2)m and m is 0 or 1. Preferably, R5 means xe2x80x94(CH2)lxe2x80x94COOA, wherein A means preferably H, or R5 means xe2x80x94(CH2)lxe2x80x94COONH2, wherein l is 0. Preferably, R4 means xe2x80x94NH2 or xe2x80x94A, wherein A preferably means H, or preferably, R4 means xe2x80x94NHR1, wherein xe2x80x94NHR1 preferably means 
and wherein R5 of xe2x80x94NHR1 preferably means 
and l is preferably 0, or R5 of xe2x80x94NHR1 preferably means 
and l is preferably 0, or R5 of xe2x80x94NHR1 means preferably (CH2)lxe2x80x94NH2 and l is preferably 0.
A further preferred embodiment of the present invention is a compound A compound of formula I wherein R1 is a group of the following formula 
wherein Z means preferably xe2x80x94(CH2)mxe2x80x94 and m is preferably 1 and wherein R4 preferably means xe2x80x94NH2, and R5 preferably means xe2x80x94(CH2)lxe2x80x94COOA, wherein l is preferably 0 and wherein A preferably means H.
A further preferred embodiment of the present invention is a compound of formula I wherein R1 is a group of the following formula 
wherein R5 preferably means xe2x80x94(CH2)lxe2x80x94COOA, wherein l is preferably 0 and A preferably means H.
A further preferred embodiment of the present invention is a compound of formula I wherein R2 means A and A preferably means xe2x80x94CH3, or wherein R2 means xe2x80x94Exe2x80x94COOH, preferably xe2x80x94CH2xe2x80x94COOH, or wherein R2 means xe2x80x94Exe2x80x94OH, preferably xe2x80x94CH2xe2x80x94OH.
A further preferred embodiment of the present invention is a compound of formula I wherein R3 is a group of the following formula 
wherein k is preferably 2.
A further preferred embodiment of the present invention is a compound of formula I wherein R3 is a group of the following formula 
A further preferred embodiment of the present invention is a compound of formula I wherein R3 is a group of the following formula 
wherein R7 is preferably a branched C1-C10-alkyl group, preferably xe2x80x94CH(CH3)2, xe2x80x94C(CH3)3, xe2x80x94CH(CH3)CH2xe2x80x94CH3 or xe2x80x94CH2xe2x80x94CH(CH3)2, and wherein R6 preferably means xe2x80x94H, xe2x80x94COOH, xe2x80x94CONH2, xe2x80x94CH2OH, xe2x80x94CON(CH3)2 or, more preferably, wherein R6 means 
wherein q is preferably 2.
A further preferred embodiment of the present invention is a compound of formula I wherein R3 is a group of the following formula 
wherein R7 preferably means xe2x80x94CH(CH(CH3)2)2 or xe2x80x94CH2C(CH3)3.